The Pirbright Institute publication directory contains details of selected publications written by our researchers.

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Flannery J, Rajko-Nenow P, Arnold H, van Weezep E, van Rijn P A, Ngeleja C, Batten C (2019)

Improved PCR diagnostics using up-to-date in silico validation: an F-gene RT-qPCR assay for the detection of all four lineages of peste des petits ruminants virus

Journal of Virological Methods 274, 113735


Peste des petits ruminants (PPR) is a globally significant disease of small ruminants caused by the peste des petits ruminants virus (PPRV) that is considered for eradication by 2030 by the United Nations Food and Agriculture Organisation (FAO). Critical to the eradication of PPR are accurate diagnostic assays. RT-qPCR assays targeting the nucleocapsid gene of PPRV have been successfully used for the diagnosis of PPR. We describe the development of an RT-qPCR assay targeting an alternative region (the fusion (F) gene) based on the most up-to-date PPRV sequence data. In silico analysis of the F-gene RT-qPCR assay performed using PCRv software indicated 98% sensitivity and 100% specificity against all PPRV sequences published in Genbank. The assay indicated the greatest in silico sensitivity in comparison to other previously published and recommended PPRV RT-qPCR assays. We evaluated the assay using strains representative of all 4 lineages in addition to samples obtained from naturally and experimentally-infected animals. The F-gene RT-qPCR assay showed 100% diagnostic specificity and demonstrated a limit of detection of 10 PPRV genome copies per µl. This RT-qPCR assay can be used in isolation or in conjunction with other assays for confirmation of PPR and should support the global efforts for eradication.

Banjara S, Shimmon G L, Dixon L K, Netherton C L, Hinds M G, Kvansakul M (2019)

Crystal structure of African swine fever virus A179L with the autophagy regulator Beclin

Viruses 11 (9), 36-37
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Subversion of programmed cell death-based host defence systems is a prominent feature of infections by large DNA viruses. African swine fever virus (ASFV) is a large DNA virus and sole member of the Asfarviridae family that harbours the B-cell lymphoma 2 or Bcl-2 homolog A179L. A179L has been shown to bind to a range of cell death-inducing host proteins, including pro-apoptotic Bcl-2 proteins as well as the autophagy regulator Beclin. Here we report the crystal structure of A179L bound to the Beclin BH3 motif. A179L engages Beclin using the same canonical ligand-binding groove that is utilized to bind to pro-apoptotic Bcl-2 proteins. The mode of binding of Beclin to A179L mirrors that of Beclin binding to human Bcl-2 and Bcl-xL as well as murine gamma-herpesvirus 68. The introduction of bulky hydrophobic residues into the A179L ligand-binding groove via site-directed mutagenesis ablates binding of Beclin to A179L, leading to a loss of the ability of A179L to modulate autophagosome formation in Vero cells during starvation. Our findings provide a mechanistic understanding for the potent autophagy inhibitory activity of A179L and serve as a platform for more detailed investigations into the role of autophagy during ASFV infection.

Aira C, Ruiz T, Dixon L, Blome S, Rueda P, Sastre P (2019)

Bead-based multiplex assay for the simultaneous detection of antibodies to African swine fever virus and classical swine fever virus

Frontiers in Veterinary Science 6, 306


African swine fever (ASF) and Classical swine fever (CSF) are both highly contagious diseases of domestic pigs and wild boar. In the last years, several cases of both diseases have been reported in the Caucasus, Russian Federation and Eastern Europe. Thus, the probability of encountering these two viruses in the same area is increasing. Since differentiation by clinical or post-mortem examination is not possible, laboratory tools for differential diagnosis are required. In the present work, we have developed a triplex bead-based assay using some of the most immunogenic antigens of each virus, for the simultaneous detection of antibodies; i.e. the VP72 and VP30 of ASF virus (ASFV) and the E2 protein of CSF virus (CSFV). The assay was firstly set up and optimized using well characterized reference serum samples specific for each pathogen. Then, a panel of 352 sera from experimentally infected animals with either ASFV or CSFV were analyzed in the multiplex assay. A collection of 253 field negative sera was also included in the study. The results of the multiplex analysis were compared to those obtained by two commercially available ELISAs for detection of antibodies against ASFV or CSFV, and considered in this study as the reference techniques. The data obtained showed values of 97.3% sensitivity and 98.3% specificity for detection of antibodies to ASFV and 95.7% of sensitivity and 99.8% specificity for detection of antibodies to CSFV. This multiplex assay allows the simultaneous and differential detection of antibodies against ASFV and CSFV, providing a valuable tool for surveillance studies. Moreover, this method is rather versatile, offering the possibility of increasing the panel of antigens from other swine diseases that could be of interest for a differential diagnosis along with ASF and CSF.

Yao Y, Ding C, Nair V (2019)

Herpesvirus of turkeys (Meleagridis herpesvirus 1) encodes a functional microRNA-221 homolog with high sequence conservation

Advances in Microbiology 9 (8), 728-736


Herpesviruses account for most of the known virus-encoded miRNAs. Herpesvirus of turkey (HVT), a non-pathogenic avian herpesvirus used as an avian vaccine and viral vector, encodes 28 mature miRNAs. This included HVT-miR-H14-3p that showed almost identical sequence to gga-miR-221, suggesting that it is pirated from the avian host. Although the functional homolog between the two miRNAs has been proposed based on the sequence similarity, the direct experimental evidence is still lacking. In this report, we provide the evidence for the first time that HVT-miR-H14-3p is indeed a gga-miR-221 homolog through modulating the expression of p27Kip1, a known target of miR-221 by binding to its 3’UTR. We also created an HVT-miR-H14-3p deletion virus and show that this miRNA is not essential for in vitro replication.

Sealy J E, Fournie G, Trang P H, Dang N H, Sadeyen J-R, Thanh T L, van Doorn H R, Bryant J E, Iqbal M (2019)

Poultry trading behaviours in Vietnamese live bird markets as risk factors for avian influenza infection in chickens

Transboundary and Emerging Diseases Early View,
Publisher’s version:


Vietnamese poultry are host to co-circulating subtypes of avian influenza viruses, including H5N1 and H9N2, which pose a great risk to poultry productivity and to human health. AIVs circulate throughout the poultry trade network in Vietnam, with live bird markets being an integral component to this network. Traders at LBMs exhibit a variety of trading practices, which may influence the transmission of AIVs. We identified trading practices that impacted on AIV prevalence in chickens marketed in northern Vietnamese LBMs. We generated sequencing data for 31 H9N2 and 2 H5N6 viruses. Viruses isolated in the same LBM or from chickens sourced from the same province were genetically closer than viruses isolated in different LBMs or from chickens sourced in different provinces. The position of a vendor in the trading network impacted on their odds of having AIV infected chickens. Being a retailer and purchasing chickens from middlemen was associated with increased odds of infection, whereas odds decreased if vendors purchased chickens directly from large farms. Odds of infection were also higher for vendors having a greater volume of ducks unsold per day. These results indicate how the spread of AIVs is influenced by the structure of the live poultry trading network.

Ren C, Xie R, Yao Y, Yu M, Chang F, Xing L, Zhang Y, Liu Y, Wang S, Farooque M, Wang Y, Qi X, Liu C, Zhang Y, Cui H, Li K, Gao L, Pan Q, Nair V, Wang X, Gao Y (2019)

MiR-125b suppression inhibits apoptosis and negatively regulates Sema4D in avian leukosis virus-transformed cells

Viruses 11 (8), 728
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Subgroup J avian leukosis virus (ALV-J), an oncogenic retrovirus, causes hemangiomas and myeloid tumors in chickens. We previously showed that miR-125b is down-regulated in ALV-J-induced tumors. This study aimed to investigate the possible role of miR-125b in ALV-J-mediated infection and tumorigenesis. Knockdown of miR-125b expression in HP45 cells reduced, whereas over-expression induced late-stage apoptosis. Bioinformatics analysis and luciferase activity assays indicate that miR-125b targets Semaphorin 4D/CD100 (Sema4D) by binding the 3'-untranslated region of messenger RNA (mRNA). Up-regulation of miR-125b in the DF1 cell line suppressed Sema4D expression, whereas miR-125 down-regulation increased Sema4D expression levels. To uncover the function of Sema4D during ALV-J infection, animal infection experiments and in vitro assays were performed and show that Sema4D mRNA levels were up-regulated in ALV-J-infected tissues and cells. Finally, functional experiments show that miR-125 down-regulation and Sema4D over-expression inhibited apoptosis in HP45 cells. These results suggest that miR-125b and its target Sema4D might play an important role in the aggressive growth of HP45 cells induced by avian leukosis viruses (ALVs). These findings improve our understanding of the underlying mechanism of ALV-J infection and tumorigenesis. 

Paton D J, Reeve R, Capozzo A V, Ludi A (2019)

Estimating the protection afforded by foot-and-mouth disease vaccines in the laboratory

Vaccine (37), 515-5524


Foot-and-mouth disease (FMD) vaccines must be carefully selected and their application closely monitored to optimise their effectiveness. This review covers serological techniques for FMD vaccine quality control, including potency testing, vaccine matching and post-vaccination monitoring. It also discusses alternative laboratory procedures, such as antigen quantification and nucleotide sequencing, and briefly compares the approaches for FMD with those for measuring protection against influenza virus, where humoral immunity is also important. Serology is widely used to predict the protection afforded by vaccines and has great practical utility but also limitations. Animals differ in their responses to vaccines and in the protective mechanisms that they develop. Antibodies have a variety of properties and tests differ in what they measure. Antibody-virus interactions may vary between virus serotypes and strains and protection may be affected by the vaccination regime and the nature and timing of field virus challenge. Finally, tests employing biological reagents are difficult to standardise, whilst cross-protection data needed for test calibration and validation are scarce. All of this is difficult to reconcile with the desire for simple and universal criteria and thresholds for evaluating vaccines and vaccination responses and means that oversimplification of test procedures and their interpretation can lead to poor predictions. A holistic approach is therefore recommended, considering multiple sources of field, experimental and laboratory data. New antibody avidity and isotype tests seem promising alternatives to evaluate cross-protective, post-vaccination serological responses, taking account of vaccine potency as well as match. After choosing appropriate serological tests or test combinations and cut-offs, results should be interpreted cautiously and in context. Since opportunities for experimental challenge studies of cross-protection are limited and the approaches incompletely reflect real life, more field studies are needed to quantify cross-protection and its correlation to in vitro measurements.

Mahapatra M, Howson E, Fowler V, Batten C, Flannery J, Selvaraj M, Parida S (2019)

Rapid detection of peste des petits ruminants virus (PPRV) nucleic acid using a novel low-cost reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for future use in nascent PPR eradication programme

Viruses 11 (8), 699
Publisher’s version:


Peste des petits ruminants (PPR) is a disease of small ruminants caused by peste des petits ruminants virus (PPRV), and is endemic in Asia, the Middle East and Africa. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) have improved the sensitivity and rapidity of diagnosing PPR. However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for PPR would improve the fast implementation of control policies, particularly when PPR has been targeted to be eradicated by 2030. Loop-mediated isothermal amplification (LAMP) assays are simple to use, rapid, and have sensitivity and specificity within the range of RT-qPCR; and can be performed in the field using disposable consumables and portable equipment. This study describes the development of a novel RT-LAMP assay for the detection of PPRV nucleic acid by targeting the N-protein gene. The RT-LAMP assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I-IV) of PPRV. The test displayed 100% concordance with RT-qPCR when considering an RT-qPCR cut-off value of CT >40. Further, the RT-LAMP assay was evaluated using experimental and outbreak samples without prior RNA extraction making it more time and cost-effective. This assay provides a solution for a pen-side, rapid and inexpensive PPR diagnostic for use in the field in nascent PPR eradication programme.

Kelly J T, Human S, Alderman J, Jobe F, Logan L, Rix T, Gonçalves-Carneiro D, Leung C, Thakur N, Birch J, Bailey D (2019)

BST2/Tetherin overexpression modulates Morbillivirus glycoprotein production to inhibit cell–cell fusion

Viruses 11 (8), 692
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The measles virus (MeV), a member of the genus Morbillivirus, is an established pathogen of humans. A key feature of morbilliviruses is their ability to spread by virus–cell and cell–cell fusion. The latter process, which leads to syncytia formation in vitro and in vivo, is driven by the viral fusion (F) and haemagglutinin (H) glycoproteins. In this study, we demonstrate that MeV glycoproteins are sensitive to inhibition by bone marrow stromal antigen 2 (BST2/Tetherin/CD317) proteins. BST2 overexpression causes a large reduction in MeV syncytia expansion. Using quantitative cell–cell fusion assays, immunolabeling, and biochemistry we further demonstrate that ectopically expressed BST2 directly inhibits MeV cell–cell fusion. This restriction is mediated by the targeting of the MeV H glycoprotein, but not other MeV proteins. Using truncation mutants, we further establish that the C-terminal glycosyl-phosphatidylinositol (GPI) anchor of BST2 is required for the restriction of MeV replication in vitro and cell–cell fusion. By extending our study to the ruminant morbillivirus peste des petits ruminants virus (PPRV) and its natural host, sheep, we also confirm this is a broad and cross-species specific phenotype. 


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